Rabbit anti c fos

After completion of the behavioral experiments, all mice were intracardially perfused with ice-cold 0.9% saline followed by 4% paraformaldehyde. Brains were coronally sectioned at 40 µm. Brain sections were processed for immunohistochemistry detecting c-Fos using rabbit anti-c-Fos (1:3000; no. AB152MI, Santa Cruz Biotechnology) and goat anti-rabbit AlexaFluor 594 (1:300; Life Technologies) primary and secondary antibodies, respectively. c-Fos expression was captured with a ×5 lens on a fluorescent-microscope/video/computer system and quantified 500 × 500 µm area placed in mPFC at the level of the anterior horns of the corpus callosum. We counted cells in sections corresponding to the tip of the optic fiber and 1 to 2 sections anteriorly before the optic fiber for all mice. c-Fos-ir cells were detected automatically and counted with ImageJ v1.51 (NIH).

In order to study possible alterations in neural activity caused by the elevated brain KYNA level, we used an indirect immunohistochemical method. 20-μm-thick free-floating sections were washed in PB, and then incubated in 1% normal donkey serum (NDS). For the detection of c-Fos-positive neurons in the striatum and in the hippocampus, sections were exposed to the primary antibody (rabbit anti c-Fos, 1:2000; Santa Cruz) overnight at 4°C, and for 2 h to the secondary antibody (Cy3 conjugated donkey anti-rabbit, 1:500; Jackson ImmunoResearch) at room temperature. Primary and secondary antibodies were diluted in 0.1 M PB containing 0.4% Triton-X100 and 1% NDS. The sections were coverslipped with antifade mounting medium (ProLong® Gold, Life Technologies). Fluorescent photomicrographs were obtained with an Olympus BX51 microscope fitted with a DP70 digital imaging system.
Changes in c-Fos protein expression occur within 30 and 90 min after certain forms of neuromodulation. A 3-h latency period was therefore interposed after vehicle or L-KYNs administrations, for the histological study.

Brains were harvested and fixed in 4% PFA (pH7.4) for a day. The brains were then dehydrated for 24 hrs in 25% sucrose solution. All sections for KI67, DCX, c-Fos and CNPase staining were cut to a thickness of 30 μm on a sliding microtome. Sections were mounted on superfrost slides and dried overnight. Subsequently, slides were incubated in 0.01 mol/L citric buffer for 20 min at 90°C, 3% H₂O₂ for 10 min, rinsed in PBS, and incubated overnight at room temperature in rabbit anti-KI67 antibody (1:4000, Vector Lab), goat anti-DCX antibody (1:250, Santa Cruz), rabbit anti-c-Fos (1:1000, Santa Cruz), or rabbit anti-CNPase (1:1000, Abcam). The next day, a standard IgG ABC kit (Vector Lab) procedure was used and the slides reacted for 5–10 min with a Sigma DAB tablet. Sections were then counterstained with cresyl violet and cover-slipped with DPX.
For BrdU staining, following a 3% H₂O₂ incubation for 10 min, slides were subsequently incubated in 2M HCL for 30 min at 37°C. Rat anti-BrdU antibody (1:250, Accurate) was applied overnight. The next day the ABC kit procedure was followed and the slides were reacted with a Sigma DAB tablet. Hippocampus cells were counted bilaterally on every eighth section through the entire rostrocaudal extent of the granule cell layer.

Recombinant mouse leptin was obtained from the National Hormone and Peptide Program (Dr. A. F. Parlow, Los Angeles, CA, USA), mouse anti-CART was a generous gift from Dr. J. T. Claussen (no. Ca6-1 F4D4; Novo Nordisk A/S, Bagsvaerd, Denmark), rabbit anti-c-Fos was from Santa Cruz Biotechnology (no. sc-52, Santa Cruz, CA, USA). Normal donkey serum (NDS), biotinylated donkey-anti-rabbit immunoglobulin (Ig)G and the cyanine² (Cy²)-conjugated donkey-anti-mouse, Cy³-conjugated donkey-anti-rabbit sera were from Jackson ImmunoResearch (West Grove, PA, USA). ABC Elite solution were purchased from Vector Laboratories (Burlingame, CA, USA). All other immunoreagents were from Sigma Chemical (St. Louis, MO, USA).